Hck activity is markedly different in untreated versus cured TpM409 cells and in resting versus stimulated bovine B cells.
(A) Kinase activity of Hck immunoprecipitated (IP) from total TpM409 extracts was determined by in vitro kinase assays. Endogenous tyrosine phosphorylation status of Hck was investigated by performing kinase assays with (+) or without (−) 10 μM Src kinase inhibitor PP2. In the absence of PP2, Hck phosphorylates the enolase substrate (middle panel). Antiphosphotyrosine Western blotting of the kinase assay products (bottom panel) shows that Hck is only poorly tyrosine phosphorylated in the absence of in vitro kinase activity. Equal loading was confirmed by Western blot using Hck-specific antibodies (top panel). (B) Hck kinase activity immunoprecipitated from 2 × 107 untreated, BW720c-cured TpM409 cells or cured TpM409 cells stimulated for 24 hours with PMA/ionomycin was determined as in panel A (p-Hck, autophosphorylated Hck; p-enolase, phosphorylated enolase). (C) Hck kinase activity immunoprecipitated from 1.5 × 108 resting B cells or the same number of B cells stimulated for 24 hours with PMA/ionomycin was determined as described in panel A. A comparable quantity of Hck is expressed in resting or stimulated B cells (top panel), whereas stimulation increases Hck kinase activity (bottom panel).
